Process for producing HMG-CoA reductase inhibitors

ABSTRACT

The present invention relates to a process for producing a compound (II-a) or a compound (II-b), each of which is a hydroxylated product of a compound represented by the formula (I-a) (hereinafter referred to as compound (I-a)): 
                 
 
wherein
         R 1  represents a hydrogen atom, a substituted or unsubstituted alkyl, or an alkali metal, and R 2  represents a substituted or unsubstituted alkyl, or a substituted or unsubstituted aryl;   or a ring-closed lactone form thereof (hereinafter referred to as compound (I-b)).
 
wherein the process comprises:
   treating the compound (I-a) or compound (I-b) in an aqueous medium comprising with a microorganism acting to hydroxylate compound (I-a) or compound (I-b), having no ability to sporulate and showing no hyphal growth, a culture of the microorganism, or a treated product of the culture, as an enzyme source; and   collecting a hydroxylated product of compound (I-a) or compound (I-b) from the aqueous medium.

TECHNICAL FIELD

The present invention relates to a process for producing a compound,which inhibits hydroxymethylglutaryl CoA (HMG-CoA) reductase and has anaction of reducing serum cholesterol.

BACKGROUND ART

A compound represented by the formula (VI-a) (hereinafter referred to ascompound (VI-a)):

wherein R¹ represents a hydrogen atom or an alkali metal, or a lactoneform of compound (VI-a) represented by the formula (VI-b) (hereinafterreferred to as compound (VI-b)):

is known to inhibit HMG-CoA reductase and exhibit an action of reducingserum cholesterol and the like (The Journal of Antibiotics, 29, 1346(1976)).

There have been several reports regarding a method for producing thecompound (VI-a) or the compound (VI-b) from a compound represented bythe formula (V-a) (hereinafter referred to as compound (V-a)):

wherein R¹ represents a hydrogen atom or an alkali metal, or the lactoneform of compound (V-a) represented by the formula (V-b) (hereinafterreferred to as compound (V-b)):

using a microorganism.

Specifically, Japanese Patent Application Laid-Open (kokai) No. 57-50894describes a method which uses filamentous fungi; both Japanese PatentApplication Laid-Open (kokai) No. 7-184670 and International PublicationWO96/40863 describe a method which uses Actinomycetes; and JapanesePatent No. 2672551 describes a method which uses recombinantActinomycetes. As is well known, however, since filamentous fungi andActinomycetes grow with filamentous form by elongating hyphae, theviscosity of the culture in a fermentor increases. This often causesshortage of oxygen in the culture, and since the culture becomesheterogeneous, reaction efficiency tends to be reduced. In order toresolve this oxygen shortage and maintain homogeneousness of theculture, the agitation rate of the fermentor should be raised, but byraising the agitation rate, hyphae are sheared and activity of themicroorganisms tends to decrease (Basic Fermentation Engineering (HakkoKogaku no Kiso) p. 169-190, P. F. Stansbury, A. Whitaker, JapanScientific Societies Press (1988)).

Furthermore, the above Actinomycetes and filamentous fungi have anability to sporulate. Since spores tend to disperse much more easilythan cells and have an ability of surviving even under conditions wherevegetative cells perish readily, these spores tend to become a source ofmicroorganism contamination in culturing and purification processes.

DISCLOSURE OF THE INVENTION

The object of the present invention is to provide an industriallyadvantageous method for producing a compound which inhibits HMG-CoAreductase and has an action of reducing the level of serum cholesteroland the like.

The present inventors have considered that if hydroxylation of compound(V-a) or compound (V-b) could be carried out with a microorganism havinghydroxylation activity, having no ability to sporulate and showing nohyphal growth, inconvenience such as the decrease of reaction efficiencydue to microorganism contamination caused by the release of sporesduring the production process or the heterogeneity of the culture causedby formation of hyphae could be avoided, and that this would beindustrially advantageous. As a result of intensive studies directed tothis object, the present inventors have accomplished the presentinvention.

Thus, the present invention relates to the following (1) to (9).

Hereinafter, in the formulas, R¹ represents a hydrogen atom, asubstituted or unsubstituted alkyl, or an alkali metal, and R²represents a substituted or unsubstituted alkyl, or a substituted orunsubstituted aryl, unless otherwise specified.

(1) A process for producing a compound (II-a) or a compound (II-b)wherein a microorganism having an activity of producing compound (II-a)or a compound (II-b) from a compound (I-a) or a compound (I-b), havingno ability to sporulate and showing no hyphal growth, a culture of saidmicroorganism, or a treated product of said culture is used as an enzymesource, and the process comprises: allowing the compound (I-a) or thecompound (I-b) to exist in an aqueous medium; allowing the compound(II-a) or the compound (II-b) to be produced and accumulated in saidaqueous medium; and collecting the compound (II-a) or the compound(II-b) from said aqueous medium, and wherein the compound (I-a) is acompound represented by the formula (I-a) (herein referred to ascompound (I-a)):

the compound (I-b) is a lactone form of compound (I-a) represented bythe compound (I-b) (herein referred to as compound (I-b)):

the compound (II-a) is a compound represented by the formula (II-a)(herein referred to as compound (II-a)):

and the compound (II-b) is a lactone form of compound (II-a) representedby the formula (II-b) (herein referred to as compound (II-b)):

(2) The process according to (1) above, wherein the compound (I-a) is acompound represented by the formula (III-a) (herein referred to ascompound (III-a)):

the compound (I-b) is a compound represented by the formula (III-b)(herein referred to as compound (III-b)):

the compound (II-a) is a compound represented by the formula (IV-a)(herein referred to as compound (IV-a)):

the compound (II-b) is a compound represented by the formula (IV-b)(herein referred to as compound (IV-b)):

(3) The process according to (1) above, wherein the compound (I-a) is acompound represented by the formula (V-a) (herein referred to ascompound (V-a)):

the compound (I-b) is a compound represented by the formula (V-b)(herein referred to as compound (V-b)):

the compound (II-a) is a compound represented by the formula (VI-a)(herein referred to as compound (VI-a)):

and; the compound (II-b) is a compound represented by the formula (VI-b)(herein referred to as compound (VI-b)):

(4) The process according to (1) above, wherein the compound (I-a) is acompound represented by the formula (VII-a) (herein referred to ascompound (VII-a)):

the compound (I-b) is a compound represented by the formula (VII-b)(herein referred to as compound (VII-b)):

the compound (II-a) is a compound represented by the formula (VIII-a)(herein referred to as compound (VIII-a)):

and, the compound (II-b) is a compound represented by the formula(VIII-b) (herein referred to as compound (VIII-b)):

(5) The process according to (1), wherein the treated product of theculture of the microorganism is a treated product selected from culturedcells; treated products such as dried cells, freeze-dried cells, cellstreated with a surfactant, cells treated with an enzyme, cells treatedby ultrasonication, cells treated by mechanical milling, cells treatedby solvent; a protein fraction of a cell; and an immobilized products ofcells or treated cells.

(6) The process according to (1) above, wherein the microorganism isselected from those belonging to the genus Mycobacterium,Corynebacterium, Brevibacterium, Rhodococcus, Gordonia, Arthrobacter,Micrococcus, Cellulomonas and Sphingomonas.

(7) The processing according to (1) above, wherein the microorganism isone selected from Mycobacterium phlei, Mycobacterium smegmatis,Mycobacterium thermoresistibile, Mycobacterium neoaurum, Mycobacteriumparafortuitum, Mycobacterium gilvum, Rhodococcus globerulus, Rhodococcusequi, Rhodococcus erythropolis, Rhodococcus rhodochrous, Rhodococcusrhodnii, Rhodococcus ruber, Rhodococcus coprophilus, Rhodococcusfascians, Gordonia amarae, Gordonia rubropertinctus, Gordoniabronchialis, Gordonia sputi, Gordonia aichiensis, Gordonia terrae,Corynebacterium glutamicum, Corynebacterium mycetoides, Corynebacteriumvariabilis, Corynebacterium ammoniagenes, Arthrobacter crystallopoietes,Arthrobacter duodecadis, Arthrobacter ramosus, Arthrobacter sulfureus,Arthrobacter aurescens, Arthrobacter citreus, Arthrobacter globiformis,Brevibacterium acetylicum, Brevibacterium linens, Brevibacteriumincertum, Brevibacterium iodinum, Micrococcus luteus, Micrococcusroseus, Cellulomonas cellulans, Cellulomonas cartae, Sphingomonaspaucimobilis, Sphingomonas adhaesiva, and Sphingomonas terrae.

(8) The process according to (1) above, wherein the microorganism is oneselected from Mycobacterium phlei JCM5865, Mycobacterium smegmatisJCM5866, Mycobacterium thermoresistibile JCM6362, Mycobacterium neoaurumJCM6365, Mycobacterium parafortuitum JCM6367, Mycobacterium gilvumJCM6395, Rhodococcus globerulus ATCC25714, Rhodococcus equi ATCC21387,Rhodococcus equi ATCC7005, Rhodococcus erythropolis ATCC4277,Rhodococcus rhodochrous ATCC21430, Rhodococcus rhodochrous ATCC13808,Rhodococcus rhodnii ATCC35071, Rhodococcus ruber JCM3205, Rhodococcuscoprophilus ATCC29080, Rhodococcus fascians ATCC12974, Rhodococcusfascians ATCC35014, Gordonia amarae ATCC27808, Gordonia rubropertinctusIFM-33, Gordonia rubropertinctus ATCC14352, Gordonia bronchialisATCC25592, Gordonia sputi ATCC29627, Gordonia aichiensis ATCC33611,Gordonia terrae ATCC25594, Corynebacterium glutamicum ATCC13032,Corynebacterium glutamicum ATCC14020, Corynebacterium glutamicumATCC19240, Corynebacterium mycetoides ATCC21134, Corynebacteriumvariabilis ATCC15753, Corynebacterium ammoniagenes ATCC6872,Arthrobacter crystallopoietes ATCC15481, Arthrobacter duodecadisATCC13347, Arthrobacter ramosus ATCC13727, Arthrobacter sulfureusATCC19098, Arthrobacter aurescens ATCC13344, Arthrobacter citreusATCC11624, Arthrobacter globiformis ATCC8010, Brevibacterium acetylicumATCC953, Brevibacterium linens ATCC19391, Brevibacterium linensATCC9172, Brevibacterium incertum ATCC8363, Brevibacterium iodinumIFO3558, Micrococcus luteus ATCC4698, Micrococcus roseus ATCC186,Cellulomonas cellulans ATCC15921, Cellulomonas cartae ATCC21681,Sphingomonas paucimobilis ATCC29837, Sphingomonas adhaesiva JCM7370, andSphingomonas terrae ATCC15098. The Institute for Fermentation (IFO),Osaka, is located at 17-85, Juso-honmachi, 2-chrome, Yodogawa-ku, Osaka532-8686, Japan. The Japan Collection of Microorganisms (JCM), RIKEN(The Institute of Physical and Chemical Research), is located at 2-1Hirosawa, Wako, Saitama 351-0198, Japan.

(9) The process according to (1) above, wherein the microorganism isGordonia sp. ATCC19067.

The present invention is described in detail below.

Examples of an enzyme source used in the present invention include: amicroorganism which has an activity of producing the above compound(II-a) or the above compound (II-b) from the above compounds (I-a) orthe above compound (I-b), having no ability to sporulate and showing nohyphal growth; a culture of said microorganism; or a treated product ofsaid culture.

Alkyl is a linear or branched alkyl containing 1 to 10 carbon atoms,preferably 1 to 6 carbon atoms, such as methyl, ethyl, propyl,isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl,hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl,2,2,4-trimethylpentyl, nonyl, decyl, and various branched chain isomersthereof.

Examples of aryl include phenyl and naphtyl.

The substituent of the substituted alkyl may be 1 to 3 identical ordifferent groups, and examples thereof include halogens, hydroxy, amino,alkoxy and aryl.

The substituent of the substituted aryl may be 1 to 3 identical ordifferent groups, and examples thereof include halogens, hydroxy, amino,alkyl and alkoxy.

The alkyl moiety of the alkoxy has the same definition as in the alkylmentioned above.

Alkali metal represents each element of lithium, sodium, potassium,rubidium, cesium or francium.

Examples of the above microorganism include microorganisms selected fromthe genus Mycobacterium, Corynebacterium, Brevibacterium, Rhodococcus,Gordonia, Arthrobacter, Micrococcus, Cellulomonas and Sphingomonas.

Specific examples include microorganisms selected from Mycobacteriumphlei, Mycobacterium smegmatis, Mycobacterium thermoresistibile,Mycobacterium neoaurum, Mycobacterium parafortuitum, Mycobacteriumgilvum, Rhodococcus globerulus, Rhodococcus equi, Rhodococcuserythropolis, Rhodococcus rhodochrous, Rhodococcus rhodnii, Rhodococcusruber, Rhodococcus coprophilus, Rhodococcus fascians, Gordonia amarae,Gordonia rubropertinctus, Gordonia bronchialis, Gordonia sputi, Gordoniaaichiensis, Gordonia terrae, Corynebacterium glutamicum, Corynebacteriummycetoides, Corynebacterium variabilis, Corynebacterium ammoniagenes,Arthrobacter crystallopoietes, Arthrobacter duodecadis, Arthrobacterramosus, Arthrobacter sulfureus, Arthrobacter aurescens, Arthrobactercitreus, Arthrobacter globiformis, Brevibacterium acetylicum,Brevibacterium linens, Brevibacterium incertum, Brevibacterium iodinum,Micrococcus luteus, Micrococcus roseus, Cellulomonas cellulans,Cellulomonas cartae, Sphingomonas paucimobilis, Sphingomonas adhaesiva,and Sphingomonas terrae.

More specifically, examples include Mycobacterium phlei JCM5865,Mycobacterium smegmatis JCM5866, Mycobacterium thermoresistibileJCM6362, Mycobacterium neoaurum JCM6365, Mycobacterium parafortuitumJCM6367, Mycobacterium gilvum JCM6395, Rhodococcus globerulus ATCC25714,Rhodococcus equi ATCC21387, Rhodococcus equi ATCC7005, Rhodococcuserythropolis ATCC4277, Rhodococcus rhodochrous ATCC21430, Rhodococcusrhodochrous ATCC13808, Rhodococcus rhodnii ATCC35071, Rhodococcus ruberJCM3205, Rhodococcus coprophilus ATCC29080, Rhodococcus fasciansATCC12974, Rhodococcus fascians ATCC35014, Gordonia amarae ATCC27808,Gordonia rubropertinctus IFM-33, Gordonia rubropertinctus ATCC14352,Gordonia bronchialis ATCC25592, Gordonia sputi ATCC29627, Gordoniaaichiensis ATCC33611, Gordonia terrae ATCC25594, Corynebacteriumglutamicum ATCC13032, Corynebacterium glutamicum ATCC14020,Corynebacterium glutamicum ATCC19240, Corynebacterium mycetoidesATCC21134, Corynebacterium variabilis ATCC15753, Corynebacteriumammoniagenes ATCC6872, Arthrobacter crystallopoietes ATCC15481,Arthrobacter duodecadis ATCC13347, Arthrobacter ramosus ATCC13727,Arthrobacter sulfureus ATCC19098, Arthrobacter aurescens ATCC13344,Arthrobacter citreus ATCC11624, Arthrobacter globiformis ATCC8010,Brevibacterium acetylicum ATCC953, Brevibacterium linens ATCC19391,Brevibacterium linens ATCC9172, Brevibacterium incertum ATCC8363,Brevibacterium iodinum IFO3558, Micrococcus luteus ATCC4698, Micrococcusroseus ATCC186, Cellulomonas cellulans ATCC15921, Cellulomonas cartaeATCC21681, Sphingomonas paucimobilis ATCC29837, Sphingomonas adhaesivaJCM7370, Sphingomonas terrae ATCC15098 and Gordonia sp. ATCC19067.

In addition, a subculture, mutant, derivative or recombinant produced bya recombinant DNA technique of any of these microorganisms can also beused.

As a medium used for the culture of the microorganism used in thepresent invention, both natural and synthetic media can be used, as longas the media contain a carbon source, a nitrogen source, inorganic saltsand the like which can be assimilated by the microorganism of thepresent invention, and can achieve an efficient culture of themicroorganism of the present invention.

Specific examples of the carbon source in a medium include carbohydratessuch as glucose, fructose, glycerol, maltose, starch and saccharose, andorganic acids such as acetic acid and citric acid and molasses.

Specific examples of the nitrogen source include ammonia; ammonium saltsof various types of inorganic acids and organic acids, such as ammoniumchloride, ammonium sulfate, ammonium acetate, ammonium nitrate andammonium phosphate; peptone, meat extract, corn steep liquor, caseinhydrolysate, soybean meal, cottonseed meal, fish meal, various types offermented microbial cells and digests thereof.

Specific examples of inorganic substances include potassiumdihydrogenphosphate, dipotassium hydrogenphosphate, magnesium phosphate,magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate,copper sulfate, and calcium carbonate.

Vitamins such as thiamin and biotin, amino acids such as glutamic acidand aspartic acid, nucleic acid-related substances such as adenine andguanine may be added, as required.

The culturing of the microorganism used in the present invention ispreferably carried out under aerobic conditions such as a shakingculture, an aeration-agitation culture or the like. Where theaeration-agitation culture is applied, it is preferred to add anappropriate amount of antifoaming agent to prevent foaming. The cultureis carried out usually at 20 to 50° C., preferably at 25 to 40° C., for6 to 120 hours. During culturing, pH is maintained at 5.0 to 10.0,preferably at 6.0 to 8.5. The pH control is carried out by usinginorganic or organic acid, an alkaline solution, urea, calciumcarbonate, ammonia, etc.

Examples of a treated product of the thus-obtained culturedmicroorganism include cultured cells; a treated product such as driedcells, freeze-dried cells, cells treated with a surfactant, cellstreated with an enzyme, cells treated by ultrasonication, cells treatedby mechanical milling, cell treated by solvent; a protein fraction ofcells; and an immobilized product of cells or treated cells.

The methods for converting compound (I-a) or compound (I-b) intocompound (II-a) or compound (II-b) may be a method of previously addingcompound (I-a) or compound (I-b) to a medium in which a microorganism isto be cultured, or may be a method of adding compound (I-a) or compound(I-b) during culturing. Further, a method of allowing an enzyme sourceto act upon compound (I-a) or compound (I-b) in an aqueous medium mayalso be used.

In a case where compound (I-a) or compound (I-b) is added to a medium inwhich a microorganism is to be cultured, 0.1 to 10 mg, preferably 0.2 to1 mg of the compound (I-a) or the compound (I-b) is added to 1 ml ofmedium at the beginning of or at some midpoint of the culture. Whencompound (I-a) or compound (I-b) is added, it may be added after it isdissolved in a solvent such as methyl alcohol or ethyl alcohol.

In a case where a method of allowing an enzyme source to act uponcompound (I-a) or compound (I-b) in an aqueous medium, the amount ofenzyme to be used depends on the specific activity of the enzyme sourceor the like. For example, when a culture of a microorganism or a treatedproduct of the culture is used as an enzyme source, 5 to 1,000 mg,preferably 10 to 400 mg of enzyme source is added per 1 mg of compound(I-a) or compound (I-b). The reaction is performed in an aqueous medium,preferably at 20 to 50° C., and particularly preferably at 25 to 40° C.The reaction period depends on the amount, specific activity, etc. ofthe enzyme source to be used, but it is usually 0.5 to 150 hours,preferably 1 to 72 hours.

Examples of an aqueous medium include water or buffers such as phosphatebuffer, HEPES (N-2 hydroxyethylpiperazine-N-ethanesulfonate) buffer andTris (tris(hydroxymethyl)aminomethane)hydrochloride buffer. An organicsolvent may be added to the above buffers, unless it inhibits reaction.Examples of organic solvent include acetone, ethyl acetate, dimethylsulfoxide, xylene, methyl alcohol, ethyl alcohol and butanol. A mixtureof an organic solvent and an aqueous medium is preferably used whencompound (I-b) is used.

According to the above production method, compound (II-b) or a mixtureof compound (II-a) and compound (II-b) can be obtained from compound(I-a).

Similarly, compound (II-b) or a mixture of compound (II-a) and compound(II-b) can be obtained from compound (I-b).

Moreover, a mixture of compound (II-a) and compound (II-b) can beobtained from a mixture of compound (I-a) and compound (I-b).

Compound (I-b) and compound (II-b) can easily be converted into compound(I-a) and compound (II-a) respectively, by a method for opening alactone ring as mentioned below. Likewise, compound (I-a) and compound(II-a) can easily be converted into compound (I-b) and compound (II-b)respectively, by a method for producing lactone as mentioned below.

Examples of a method for opening a lactone ring include a method whichcomprises dissolving compound (I-b) or compound (II-b) in an aqueousmedium and adding thereto an acid or alkali. Examples of the aqueousmedium include water and an aqueous solution containing salts, whichdoes not inhibit the reaction, such as phosphate buffer, Tris buffer andthe like. The above aqueous solution may contain an organic solvent suchas methanol, ethanol, ethyl acetate and the like in a concentrationwhich does not inhibit the reaction. Examples of acid include aceticacid, hydrochloric acid and sulfuric acid, and examples of alkaliinclude sodium hydroxide, potassium hydroxide and ammonia.

Examples of a method for producing lactone include a method whichcomprises dissolving compound (I-a) or compound (II-a) in a non-aqueoussolvent and adding thereto an acid or base catalyst. As long as thenon-aqueous solvent is an organic solvent which does not substantiallycontain water and is capable of dissolving compound (I-a) or compound(II-a), any type of non-aqueous solvent can be used.

Examples of non-aqueous solvents include dichloromethane and ethylacetate. As a catalyst, any catalyst can be used, as long as itcatalyzes lactonization and does not show any actions other thanlactonization on a substrate or a reaction product. Examples of theabove catalyst include trifluoroacetic acid and p-toluenesulfonic acid.Reaction temperature is not particularly limited, but is preferably 0 to100° C., and is more preferably 20 to 80° C.

After completion of the reaction, compound (II-a) or compound (II-b) canbe collected from the above solution by ordinary methods used in thefield of organic synthetic chemistry such as extraction with organicsolvents, crystallization, thin-layer chromatography, high performanceliquid chromatography, etc.

As a method for detecting and quantifying the compound (II-a) or thecompound (II-b) obtained by the present invention, any method can beused, as long as the detection or quantification of compound (II-a)and/or compound (II-b) can be performed. Examples thereof include¹³C-NMR spectroscopy, ¹H-NMR spectroscopy, mass spectroscopy, highperformance liquid chromatography (HPLC) etc.

There may be stereoisomers such as optical isomers for some compoundsamong compound (I-a), compound (I-b), compound (II-a) and compound(II-b). The present invention covers all possible isomers and mixturesthereof including these stereoisomers.

As compound (I-a), compound (III-a) is preferably, compound (V-a) ismore preferable, and compound (VII-a) is particularly preferable.

As compound (I-b), compound (III-b) is preferable, compound (V-b) ismore preferable, and compound (VII-b) is particularly preferable.

As compound (II-a), compound (IV-a) is preferable, compound (VI-a) ismore preferable, and compound (VIII-a) is particularly preferable.

As compound (II-b), compound (IV-b) is preferable, compound (VI-b) ismore preferable, and compound (VII-b) is particularly preferable.

The examples of the present invention is described below, but thepresent invention is not limited to these examples.

THE BEST MOST FOR CARRYING OUT THE INVENTION EXAMPLE 1

100 mg of compound (VII-b) (produced by Sigma) was dissolved in 9.5 mlof methanol, and 0.5 ml of 1 mol/l sodium hydroxide was added. Themixture was stirred at room temperature for 1 hour. The obtainedreaction solution was dried to be solidified, and was dissolved byadding 5 ml of deionized water, followed by adjusting pH to around 6.5to 7.5 with about 0.1 ml of 1 mol/l hydrochloric acid. Then, 4.9 ml ofdeionized water was added to the mixture to obtain 10 ml of compound(VII-a), whose final concentration was 10 mg/ml (a compound wherein, informula (VII-a), R¹ is sodium).

Various types of microorganisms shown in Tables 1 and 2 wereindependently plated onto an agar medium (1% peptone (produced byKyokuto Pharmaceutical Industrial Co., Ltd.), 0.7% meat extract(produced by Kyokuto Pharmaceutical Industrial Co., Ltd.), 0.3% NaCl,0.2% yeast extract (produced by Nihon Pharmaceutical Co., Ltd.), 2%bacto agar (produced by Difco), adjusted to pH7.2 with 1 mol/l sodiumhydroxide), then cultured for 3 days at each temperature shown in Tables1 and 2. An inoculating loop of each of the strains which grew on theagar medium was inoculated into a test tube containing 3 ml of LB medium(1% bacto tryptone (produced by Difco), 0.5% bacto yeast extract(produced by Difco), adjusted to pH7.2 with 1 mol/l sodium hydroxide).This tube was then subjected to shaking culture for 24 hours at eachtemperature shown in Tables 1 and 2. After culturing, 0.25 ml of theculture was inoculated in test tubes containing 5 ml of TB medium (1.4%bacto tryptone (produced by Difco), 2.4% bacto yeast extract (producedby Difco), 0.231% KH₂PO₄, 1.251% K₂HPO₄, adjusted to pH7.4 with 1 mol/lsodium hydroxide). The tubes were then subjected to shaking culture for24 hours at each temperature shown in Tables 1 and 2. After 24 hours,the above-obtained compound (VII-a) was added to each of test tubes in athe final concentration of 0.4 mg/ml, and then reaction was performedwith shaking at each temperature shown in Tables 1 and 2 for 48 hours.

After completion of the reaction, the reaction solution was adjusted topH3.5 with acetic acid. 1 ml of ethyl acetate was added to 0.5 ml ofthis reaction solution followed by shaking for 1 hour. After shaking,the reaction solution was separated into 2 layers by centrifugation at3,000 rpm for 5 minutes, then the upper ethyl acetate layer wascollected. The solvent was removed with a centrifugal evaporator, andthe residue was dissolved in 0.5 ml of methanol. Using a portion of thismethanol solution, HPLC analysis was carried out (Column: Inertsil ODS-2(5 μm, 4×250 mm, produced by GL Science), Column temperature: 60° C.,Mobile phase: acetonitrile:water:phosphoric acid=55:45:0.05, Flow rate:0.9 ml/min, Detection wavelength: 237 nm), to detect and quantifycompound (VIII-a) (a compound wherein, in formula (VIII-a), R¹ issodium). The results are shown in Tables 1 and 2.

TABLE 1 Culturing Compound Temperature Strain (VIII-a) mg/l (° C.)Mycobacterium phlei JCM 5865 1.6 37 Mycobacterium smegmatis JCM 5866 0.437 Mycobacterium JCM 6362 9.1 37 thermoresistibile Mycobacteriumneoaurum JCM 6365 3.7 37 Mycobacterium parafortuitum JCM 6367 7.4 37Mycobacterium gilvum JCM 6395 9.6 37 Rhodococcus globerulus ATCC257144.9 28 Rhodococcus equi ATCC21387 2.5 30 Rhodococcus erythropolisATCC4277 1.4 30 Rhodococcus rhodochrous ATCC21430 4.9 30 Rhodococcusequi ATCC7005 1.4 30 Rhodococcus rhodochrous ATCC13808 4.7 28Rhodococcus rhodnii ATCC35071 0.4 28 Rhodococcus ruber JCM 3205 0.6 28Rhodococcus coprophilus ATCC29080 5.6 28 Rhodococcus fascians ATCC129741.3 28 Rhodococcus fascians ATCC35014 5.2 30 Gordona amarae ATCC278081.2 30 Gordona rubropertinctus IFM-33 2.5 30 Gordona bronchialisATCC25592 0.9 28 Gordona rubropertinctus ATCC14352 0.7 28 Gordona sputiATCC29627 0.3 28 Gordona aichiensis ATCC33611 0.6 28 Gordona sp.ATCC19067 4.0 30 Gordona terrae ATCC25594 0.3 28

TABLE 2 Culturing Compound Temperature Strain (VIII-a) mg/l (° C.)Corynebacterium glutamicum ATCC13032 1.1 30 Corynebacterium glutamicumATCC14020 0.7 30 Corynebacterium glutamicum ATCC19240 1.0 30Corynebacterium mycetoides ATCC21134 0.3 30 Corynebacterium variabilisATCC15753 1.7 30 Corynebacterium ATCC6872 0.6 30 ammoniagenesArthrobacter crystallopoietes ATCC15481 0.5 30 Arthrobacter duodecadisATCC13347 0.7 30 Arthrobacter ramosus ATCC13727 2.2 30 Arthrobactersulfureus ATCC19098 1.1 30 Arthrobacter aurescens ATCC13344 1.3 30Arthrobacter citreus ATCC11624 1.2 30 Arthrobacter globiformis ATCC80100.3 30 Brevibacterium acetylicum ATCC953 0.4 30 Brevibacterium linensATCC19391 0.5 30 Brevibacterium linens ATCC9172 0.6 30 Brevibacteriumincertum ATCC8363 0.5 30 Brevibacterium iodinum IFO3558 0.8 30Micrococcus luteus ATCC4698 0.5 30 Micrococcus roseus ATCC186 0.4 30Cellulomonas cellulans ATCC15921 0.7 30 Cellulomonas cartae ATCC216810.7 30 Sphingomonas paucimobilis ATCC29837 3.4 30 Sphingomonas adhaesivaJCM 7370 2.7 37 Sphingomonas terrae ATCC15098 3.1 30

EXAMPLE 2

Mycobacterium gilvum JCM 6395 strain was plated onto the same agarmedium as in Example 1 and was cultured at 37° C. for 3 days. The strainwhich grew on the agar medium was inoculated into 4 test tubes eachcontaining 3 ml of LB medium, followed by shaking culture at 37° C. for24 hours. 1.25 ml of each of the cultures was inoculated into eight300-ml Erlenmeyer flasks containing 25 ml of TB medium, followed byshaking culture at 37° C. After 24 hours, compound (VII-a) prepared asin Example 1 (a compound wherein, in formula (VII-a), R¹ is sodium) wasadded in the final concentration of 0.4 mg/ml, and the mixture wasshaken at 37° C. for 48 hours. After completion of the reaction, theculture was centrifuged at 3,000 rpm at 4° C. for 10 minutes to collectthe supernatant. The pH of this supernatant was adjusted to 3.5 withacetic acid. After 400 ml of ethyl acetate was added thereto, themixture was shaken at 30° C. for 1 hour. After leaving to stand,supernatant was collected. The same operation was repeated to theaqueous lower layer, then the obtained ethyl acetate layer was combinedwith the aforementioned supernatant. After 100 ml of saturated salinesolution was added to this ethyl acetate layer, the mixture was shaken,and supernatant was collected.

Next, 5 g of anhydrous Na₂SO₄ was added to this supernatant and themixture was left at room temperature for 15 minutes. Then, ethyl acetatewas evaporated under reduced pressure so that the mixture wassolidified. The obtained residue was dissolved in 5 ml of deionizedwater, and pH was adjusted to 9.0 with sodium hydroxide, followed bypassing the solution through a 50 ml HP-20 column (25×100 mm, producedby Mitsubishi Chemical Corp.) After washing the column with 150 ml ofdeionized water, elution was carried out in a stepwise manner with 100ml of acetone solutions each of which contains 20%, 30% and 40% acetone.The collected fractions were subjected to the same HPLC analysis as inExample 1, thereby recovering a fraction containing compound (VIII-a).Acetonitrile was removed from this fraction under reduced pressure, thenpH of the solution was adjusted to 3.0 with 1 mol/l hydrochloric acid.After 360 ml of ethyl acetate was added to this solution, the mixturewas shaken. After leaving to stand, supernatant was collected. After 90ml of saturated saline solution was added to this supernatant, themixture was shaken, and left to stand, and the supernatant wascollected.

Subsequently, 4.5 g of anhydrous Na₂SO₄ was added to this supernatantand the mixture was left at room temperature for 15 minutes followed byevaporating to dryness under reduced pressure. The obtained driedresidue was dissolved in dichloromethane and lactonized by adding 1%trifluoroacetic acid. This reaction product was fractionated withpreparative TLC (Silica gel plate: No. 1.05744 (200×200 mm, thickness:0.5 mm, produced by Merck), development solvent: ethyl acetate,color-development solution: 12.5% phosphomolybdic acid-1% cerium/10%sulfuric acid solution), thereby obtaining 0.8 mg of compound (VIII-b).The results of mass spectrum and ¹H-NMR spectrum analyses of theobtained compound (VIII-b) are as follows.

Mass Spectrum

Applying JMS-HX/HX110A mass spectrometer (manufactured by NIHON DENSHILtd.), the measurement was done in a positive mode using m-nitrobenzylalcohol as a matrix. As a result, a pseudoion peak ([M+H]⁺) was obtainedat m/z 407, and the actual measurement value matched with the valueexpected from the structure and molecular weight (406) of compound(II-b).

¹H-NMR Spectrum

Applying type JNM-α400 spectrometer (manufactured by NIHON DENSHI Ltd.),the measurement was done at 400 MHz in duetero chloroform, using TMS asan internal standard. The results are shown below. The spectrum datawere consistent with the known data regarding compound (VIII-b) (SankoResearch Laboratories Annual Report, 37, 147 (1985).

δ ppm (CDCl₃): 6.01 (1H, d, J=9.5 Hz), 5.89 (1H, dd, J=9.5, 5.9 Hz),5.58 (1H, m), 5.41 (1H, m), 4.60 (1H, ddd, J=10.6, 7.3, 5.4, 2.8 Hz),4.40 (1H, m), 4.38 (1H, m), 2.74 (1H, dd, J=13.1, 6.0, 4.8, 1.5 Hz),2.40 (1H, m), 2.36 (1H, m), 2.34 (1H, m), 1.95 (1H, dddd, J=14.4, 3.7,2.9, 1.7 Hz), 1.86 (1H, dddd, J=12.5, 12.3, 7.3, 4.3 Hz), 1.69 (1H, m),1.68 (1H, m), 1.64 (1H, m), 1.57 (1H, m), 1.5-1.4 (2H, m), 1.43 (1h, m),1.30 (1H, m), 1.12 (3H, d, J=6.8 Hz), 0.91 (3H, d, J=7.1 Hz), 0.89 (3H,t, J=7.4 Hz).

Industrial Applicability

According to the present invention, it becomes possible to efficientlyproduce a compound, which inhibits HMG-CoA reductase and has an actionof reducing the level of serum cholesterol.

1. A process for producing a compound (II-a) or a compound (II-b) wherein a microorganism having an activity of producing compound (II-a) or a compound (II-b) from a compound (I-a) or a compound (I-b), selected from the group consisting of those belonging to the genus Mycobacterium, Corynebacterium, Brevibacterium, Rhodococcus, Gordonia, Arthrobacter, Micrococcus, Cellulomonas and Sphingomonas, a culture of said microorganism, or a treated product of said culture used as an enzyme source, the process comprising: allowing the compound (I-a) or the compound (I-b) to exist in an aqueous medium; allowing the compound (II-a) or the compound (II-b) to be produced and accumulated to said aqueous medium; and collecting the compound (II-a) or the compound (II-b) from said aqueous medium, and wherein the compound (I-a) is a compound represented by the formula (I-a):

 wherein R¹ represents a hydrogen atom, a substituted or unsubstituted alkyl, or an alkali metal, and R² represents a substituted or unsubstituted alkyl, or a substituted or unsubstituted aryl; the compound (I-b) is a lactone form of compound (I-a) represented by the formula (I-b):

 wherein R² has the same definition as the above; the compound (II-a) is a compound represented by the formula (II-a):

 wherein R¹ and R² have the same definitions as the above; and the compound (II-b) is a lactone form of compound (II-a) represented by the formula (II-b):

 wherein R² has the same definition as the above; and wherein the microorganism is one selected from Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium thermoresistibile, Mycobacterium neoaurum, Mycobacterium parafortuitum, Mycobacterium gilvum, Rhodococcus globerulus, Rhodococcus equi, Rhodococcus erythropolis, Rhodococcus rhodochrous, Rhodococcus rhodnii, Rhodococcus ruber, Rhodococcus coprophilus, Rhodococcus fascians, Gordonia amarae, Gordonia bronchialis, Gordonia aichiensis, Gordonia terrae, Gordonia rubropertinctus, Gordonia sputi, Corynebacterium glutamicum, Corynebacterium mycetoides, Corynebacterium variabilis, Corynebacterium ammoniagenes, Arthrobacter crystallopoietes, Arthrobacter duodecadis, Arthrobacter ramosus, Arthrobacter sulfureus, Arthrobacter aurescens, Arthrobacter citreus, Arthrobacter globiformis, Brevibacterium linens, Brevibacterium iodinum, Micrococcus luteus, Micrococcus roseus, Cellulomonas cellulans, Cellulomonas cartae, Sphingomonas paucimobilis, Sphingomonas adhaesiva, and Sphingomonas terrae.
 2. The process according to claim 1, wherein the compound (I-a) is a compound represented by the formula (III-a):

 wherein R¹ represents a hydrogen atom, a substituted or unsubstituted alkyl, or an alkali metal, and R² represents a substituted or unsubstituted alkyl, or a substituted or unsubstituted aryl; the compound (I-b) is a compound represented by the formula (III-b):

 wherein R² has the same definition as the above; the compound (II-a) is a compound represented by the formula (IV-a):

 wherein R¹ and R² have the same definitions as the above; and the compound (II-b) is a compound represented by the formula (IV-b):

 wherein R² has the same definition as the above.
 3. The process according to claim 1, wherein the compound (I-a) is a compound represented by the formula (V-a):

 wherein R¹ represents a hydrogen atom, a substituted or unsubstituted alkyl, or an alkali metal; the compound (I-b) is a compound represented by the formula (V-b);

the compound (II-a) is a compound represented by the formula (VI-a):

 wherein R¹ has the same definition as the above; and the compound (II-b) is a compound represented by the formula (VI-b):


4. The process according to claim 1, wherein the compound (I-a) is a compound represented by the formula (VII-a:

 wherein R¹ represents a hydrogen atom, a substituted or unsubstituted alkyl, or an alkali metal; the compound (I-b) is a compound represented by the formula (VII-b):

the compound (II-a) is a compound represented by the formula (VIII-a):

 wherein R¹ has the same definition as the above; and the compound (II-b) is a compound represented by the formula (VIII-b):


5. The process according to claim 1, wherein the treated product of the culture of the microorganism is a treated product selected from dried cells, freeze-dried cells, cells treated with a surfactant, cells treated with an enzyme, cells treated by ultrasonication, cells treated by mechanical milling, cells treated by solvent; and a protein fraction of a cell.
 6. The process according to claim 1, wherein the microorganism is one selected from Mycobacterium phlei JCM5865, Mycobacterium smegmatis JCM5866, Mycobacterium thermoresistibile JCM6362, Mycobacterium neoaurum JCM6365, Mycobacterium parafortuitum JCM6367, Mycobacterium gilvum JCM6395, Rhodococcus globerulus ATCC25714, Rhodococcus equi ATCC21387, Rhodococcus equi ATCC7005, Rhodococcus erythropolis ATCC4277, Rhodococcus rhodochrous ATCC21430, Rhodococcus rhodochrous ATCC13808, Rhodococcus rhodnii ATCC35071, Rhodococcus ruber JCM3205, Rhodococcus coprophilus ATCC29080, Rhodococcus fascians ATCC12974, Rhodococcus fascians ATCC35014, Gordonia amarae ATCC27808, Gordonia rubropertinctus ATCC14352, Gordonia bronchialis ATCC25592, Gordonia sputi ATCC29627, Gordonia aichiensis ATCC33611, Gordonia terrae ATCC25594, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14020, Corynebacterium glutamicum ATCC19240, Corynebacterium mycetoides ATCC21134, Corynebacterium variabilis ATCC15753, Corynebacterium ammoniagenes ATCC6872, Arthrobacter crystallopoietes ATCC15481, Arthrobacter duodecadis ATCC13347, Arthrobacter ramosus ATCC13727, Arthrobacter sulfureus ATCC19098, Arthrobacter aurescens ATCC13344, Arthrobacter citreus ATCC11624, Arthrobacter globiformis ATCC8010, Brevibacterium linens ATCC19391, Brevibacterium linens ATCC9172, Brevibacterium iodinum IFO3558, Micrococcus luteus ATCC4698, Micrococcus roseus ATCC186, Cellulomonas cellulans ATCC15921, Cellulomonas cartae ATCC21681, Sphingomonas paucimobilis ATCC29837, and Sphingomonas adhaesiva JCM7370.
 7. The process according to claim 1, wherein the microorganism is Rhodococcus rhodochrous, sp. ATCC19067. 